This invention relates to improved countercurrent chromatography systems and more particularly to multi-column planet centrifuge type countercurrent chromatography systems.
Countercurrent chromatography is the generic designation for a family of liquid-liquid partition methods that do not employ a solid supporting matrix. Thus, countercurrent chromatography offers advantages over other chromatographic methods in that it is free from all complications arising from the use of solid supports, such as excessive adsorptive loss and denaturation of samples, contamination, tailing of solute peaks, etc. However, historically the challenge for countercurrent chromatography has been to achieve partition efficiencies that are comparable to methods which rely upon solid supports.
A countercurrent chromatograph (CCC) is a device which employs two liquid phases wherein one phase is held stationary in a column (usually a coil of tubing) by gravitational or centrifugal means, while a second immiscible liquid is passed through the first. Components of a sample introduced in the stream (usually injected in a small volume) are separated according to their partition coefficients. Partition coefficients are defined herein as the ratio of solute concentration in the stationary phase to the mobile phase. Within the generic class of CCCs, is a group of CCCs referred to as Coil-Planet Centrifuges. These have the common characteristic that the stationary phase of the solvent pair is retained in the column by centrifugal means as the column revolves rapidly about a central axis. Highly efficient mixing is then achieved by further rotating the column about its own axis in a planetary motion. As a sample solution is introduced into the column, the solutes will migrate through the column at different rates according to their partition coefficients, all within the pulsating centrifugal force field. The column's coil may be wound in a "multi-layer" configuration which creates gradients within the centrifugal force field that allow faster flow rates and larger volumes of the stationary phase to be retained in the column. This type is referred to as a Multilayer Coil-Planet Centrifuge. An example of this type of CCC is disclosed in U.S. Pat. No. 4,430,216 to Ito which is hereby incorporated by reference. Another type of CCC is a "Horizontal Flow-Through Coil-Planet Centrifuge." This differs from a Multilayer Coil-Planet Centrifuge in that the Horizontal Flow-Through Coil-Planet Centrifuge employs eccentrically mounted columns. An example of this type is disclosed in U.S. Pat. No. 4,228,950 to Ito, which is incorporated by reference. These CCCs can both decrease separation time and increase resolution relative to the performance achieved in earlier art.
Techniques of chromatography may be broadly divided into preparative and analytical applications. Preparative methods relate to the purification of mixtures so as to isolate a visible amount of one or more components of the original mixture. The meaning of a visible quantity depends to some extent on the nature of the separation problem and the attitude of the individual practitioner, but generally ranges from a milligram to a gram or even larger quantities. Typically, preparative chromatography is employed in those instances where a quantity of purified substance is required for further study. Isolation of substances in the several milligram range is sometimes referred to as semi-preparative chromatography.
In analytical chromatography, the components of a mixture are simply detected in the effluent stream of a chromatograph by a suitable monitor, such as a uv light absorbing detector. The effluent is discarded with no attempt being made to save the fractions containing the isolated components. The separation is usually recorded as a graph of detector response versus effluent volume, or alternatively simply as time since flow rate is usually constant, i.e. a chromatogram. The chromatogram indicates the number of detectable components in a mixture and provides information on their chromatographic properties.
In CCC, preparative columns or coils are typically 300 ml or larger in volume while analytical columns are usually 30 ml or less. Both types are usually made of tubing with internal diameters of at least 1.6 mm for preparative columns whereas analytical columns usually have inside diameters of less than 1.07 mm.
The multi-layer type coil planet centrifuge is presently one of the most efficient CCCs in terms of good sample capacity, typically 1 gram for a 300 ml column, good resolution (separation) of sample components and fast separation times. However, to perform both preparative and analytical separations the options are either purchasing separate devices at considerable expense, or alternatively, utilizing a convertible type CCC. Multi-layer coil planet centrifuges are available in the preparative and analytical range, and they allow columns to be changed so as to accommodate a range of samples with the same instrument. However, they require the time consuming task of rebalancing since separation columns of different size vary appreciably in weight.
Countercurrent chromatography is still primarily a preparative system for purification of samples of about one gram. CCC systems have the added disadvantage of requiring several hours to run and do not offer the convenience of smaller analytical columns which permit characterizations of solvent systems in a matter of minutes.
In U.S. Pat. No. 4,228,950, Ito teaches that two columns can be placed in a coil planet centrifuge on separate axes as shown in his FIG. 1. The flow tubes from these columns can be led independently to the apparatus exterior, where they can be connected together to increase column capacity, as shown in his FIG. 3 and FIG. 5B. However, it should be noted that these columns each operate in a different manner and are subject to quite different force fields.
Column II in his FIG. 1 undergoes what may be called a "Ferris Wheel" motion. Analogous to the car in a Ferris Wheel, the column holder does not rotate on its own axis in the course of its orbit around the central axis. This motion is induced using a stationary solar pulley connected with a belt to a pulley of equal diameter on the column holder. In contrast, column I in his FIG. 1 undergoes a tumbling car motion, in which the Ferris Wheel car would be tumbled forward twice in the course of each orbit around the central axis. This is induced by using a stationary sun gear coupled to a gear of equal diameter attached to the column holder.
I have found that columns eccentrically mounted so they do not rotate on the axis they are mounted on are less conducive to stationary phase retention and exhibit generally poor separations.
Accordingly, it would be highly desirable to have an efficient and economical means for conducting countercurrent chromatography with a multiplicity of columns wound on a single axis which can provide both analytical and preparative capability with high resolution.